Recombinase-Based System for Expression of Foreign Proteins Using Adenovirus Vectors

Abstract

In the present invention, viruses, plasmids or both are constructed which contain viral DNA and lox sites positioned such that site-specific recombination between lox sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system.

Description

CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of copending parent application Ser. No. 09 / 909,414, filed Jul. 17, 2001, which is continuation of parent application Ser. No. 09 / 263,650, filed on Mar. 5, 1999 (U.S. Pat. No. 6,379,943).FIELD OF THE INVENTION [0002] The present invention relates to methods for efficient and reliable construction of adenovirus vectors that contain and express foreign DNA and are useful for gene transfer into mammalian cells, for vaccines and for gene therapy. The vector system described herein is an improvement and modification of the pBHG system, described in U.S. Pat. No. 6,140,087 (Graham et al.), a foreign equivalent of which published as WO95 / 00655, both of which are hereby incorporated by reference. BACKGROUND OF THE INVENTION [0003] As taught in U.S. Pat. No. 6,140,087, adenoviruses (Ads) can be used as mammalian cell expression vectors, with excellent potential as live recombinant viral vaccines, as transducing ...

AI Technical Summary

Benefits of technology

[0009] In the present invention, viruses, plasmids or both are constructed which contain viral DNA and lox P sites positioned such that site-specific recombination between lox P sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Such cells (293Cre cells) have been described by Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. and Graham, F. L. “A new helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal,” Proc. Natl. Acad. Sci. U.S. 93: 13565-13570, 1996, by Chen, L., Anton, M. and Graham, F. L., “Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre,” Somat. Cell and Molec. Genet. 22: 477-488, 1996, in U.S. patent application Ser. No. 08 / 473,168, and in PCT publication WO96 / 40955, hereby incorporated by reference for this purpose. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing a contaminating parental adenovirus, with the attendant problems for removal thereof.

[0011] In a further embodiment of this invention, DNA-TP complexes are utilized to combine the high efficiency of Cre-lox recombination with the high infectivity of DNA-TP. While the rescue of infectious virus via Cre mediated recombination is surprisingly efficient compared to homologous recombination, and is more than adequate to produce viral vectors and to introduce mutations into the viral genome for most applications, there may be certain applications for which even higher efficiencies are desirable or necessary. It is known by those skilled in the art that the infectivity of adenovirus DNA is up to 100 fold higher if the virion DNA is extracted and purified by methods that leave intact the terminal protein (TP) that is normally linked to the 5′ end of each strand of the duplex Ad DNA molecule (Sharp P A, Moore C, Haverty J L, “The infectivity of adenovirus 5 DNA-protein complex,” Virology 1976 December; 75(2):442-456, Chinnadurai G, Chinnadurai S, Green M, “Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex.” J Virol 1978 April; 26(1):195-199). For rescue of cassettes, the two plasmid system is more than sufficiently efficient, especially with the approximately 30-fold enhancement demonstrated herein for Cre-lox mediated recombination over homologous recombination, and consequently would be preferred for most purposes. However, there may be times when even higher efficiencies are required, as when, for example, one wishes to develop a library of fibre mutations (a large number of different viruses—the more the better). Then the chore of preparing DNA-TP might be worthwhile and could be accomplished by those skilled in the art. Thus, an aspect of the present invention includes the combination of the Cre-lox recombination with the high specific infectivity of adenoviral DNA-TP complexes.

[0012] Therefore, it is an object of the present invention to provide a highly efficient, reliable, and simple method for isolation of viral vectors based on site-specific recombination catalysed by a site-specific recombinase, such as but not limited to the Cre recombinase, rather than relying on homologous recombination, which depends on normal cellular recombinase pathways.

[0013] It is a further object of this invention to use Cre-lox-mediated recombination and known two plasmid vector production systems to provide a simple method for introducing mutations or other modifications of viral genes into any desired location in the viral genome.

[0014] It is a further object of this invention to provide a simple and useful system by which adenovirus cloning vectors may be developed.

[0016] A further object of this invention is to provide a system whereby the high-efficiency of Cre-lox recombination is combined with enhanced infectivity achieved when adenovirus-TP complexes are utilized.

Problems solved by technology

A disadvantage of this method is the need to prepare purified viral DNA.

In addition, such methods typically result in the presence of contaminating parental virus in the resulting vector preparations, such as when 100% of the viral DNA is not cleaved, or when the two viral DNA fragments produced by restriction cleavage are rejoined.

However, as Cre action is not 100% efficient, the resulting virus preparations remain contaminated with parental virus, and must be passaged in 293Cre cells to eliminate the contaminating parental virus.

A further disadvantage of this method is that it requires use of an infectious virus or DNA extracted from a virus as one of the starting materials, and is thus less attractive for commercial distribution than kits containing only bacterial plasmid DNA.

Such recombinant virus has the propensity to overgrow the original vector, leading to contamination of subsequent vector preparations with non-attenuated E1 expressing Ads.

However, although this currently available system has proven utility and is widely used, the efficiency of virus production by homologous recombination can be low and variable, and the system cannot always be used easily by those not skilled in the art.

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